Methods of Preventing or Reducing Photoreceptor Cell Death

ABSTRACT

The invention provides compositions and methods for preventing or reducing photoreceptor cell death. The invention further provides compositions and methods for treating, preventing, or alleviating symptoms of retinal detachment.

GOVERNMENT SUPPORT

This invention was made with Government support under R01EY022084awarded by National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the field of ophthalmology.

BACKGROUND OF THE INVENTION

Retinal detachment (RD) occurs in various retinal disorders, includingretinal tears, age-related macular degeneration and diabeticretinopathy, as well as a number of clinical manifestations such astractional, rhegmatogenous and exudative RD (Zacks, D. N. et al., InvestOphthalmol Vis Sci, 2006, 47:1691-1695). In patients with sustained RD,progressive visual decline due to photoreceptor cell death is common andleads to a significant decrease in visual acuity (Day S. et al., Am JOphthalmol, 2010, 150:338-345; and Rowe J. A. et al., Opthalmology,1999, 106:154-159). While numerous pathological changes occur in thedetached retina, studies in human patient samples and in animal modelshave shown that photoreceptor cell death is induced as early as 12 hoursand peaks at around 2-3 days after RD (Yu, J. et al., Invest OphthalmolVis Sci, 2012, 53:8146-8153). Untreated retinal detachment results inpermanent vision loss, and frequently results in blindness.

Currently, the only treatment available for retinal detachment is toreattach the retina by surgical procedures. However, surgery isextremely invasive and fraught with risks such as infection, bleeding,increases in intraocular pressure, and cataract. Moreover, surgery isnot always effective for reattaching the retina and/or restoring normalvision. Many patients fail to recover any lost vision. Thus, thereexists an urgent need for alternative treatments for retinal detachment,for preserving vision and/or preventing photoreceptor cell deathassociated with retinal detachment.

SUMMARY OF THE INVENTION

The invention is based on the surprising discovery that photoreceptordegeneration correlates with increased activity of components of thealternative complement pathway. Furthermore, ablation of alternativecomplement pathway components protects photoreceptor cells from celldeath.

Accordingly, compositions for preserving vision, reducing vision loss,and/or inhibiting or reducing photoreceptor cell death in a subjectcomprise an agent that inhibits or reduces complement pathway activityare described herein. The complement pathway is the alternativecomplement pathway or the lectin complement pathway. The agent comprisesa small molecule, a polynucleotide, a polypeptide, an antibody or anantibody fragment with means to inhibit or reduce the transcription,transcript stability, modification, localization, secretion, or functionof a polynucleotide or polypeptide encoding a component of thealternative or lectin complement pathway. For example, the agentcomprises a serine protease inhibitor, a soluble form of a complementreceptor, a humanized monoclonal antibody or antibody fragment, acomplement component inhibitor, a nucleic acid expression vectorencoding an anti-complement agent, a modified complement receptor or ananaphylatoxin receptor antagonist.

Preferably, the agent inhibits or reduces the activity of at least onecomponent of the complement pathway, e.g., the agent inhibits binding ofone component to another component of the pathway. Preferably, thecomplement pathway is the alternative complement pathway. Alternatively,the complement pathway is the lectin complement pathway. The agentinhibits or reduces the activity of at least one component of thealternative or lectin pathway complement pathway. Components of thealternative complement pathway include factor B (Fb), C3, properdin(Factor p), factor Ba, factor Bb, factor D, C2, C2a, C3, C3a, C5, C5a,C6, C7, C8, C9, and C5b-9. For example, the inhibitory agent is specificfor or binds to a component of the alternate complement pathway asdescribed above. Components of the lectin complement pathway includeMASP-1, MASP-2, MASP-3, Map19, Map44, C4, C4a, C4b, C2, C2a and C2b. Ina preferred embodiment, the agent specifically binds to the complementpathway component to modulate the transcription, transcript stability,modification, localization, secretion, or function of the component.

For example, the agent that inhibits or reduces the activity of at leastone component of the complement pathway is an antibody or an antibodyfragment. The antibody or antibody fragment specifically binds to analternative complement component, such as factor B, C3, properdin(Factor p), factor Ba, factor Bb, factor D, C2, C2a, C3, C3a, C3b C5,C5a, C5b, C6, C7, C8, C9, or C5b-9. The antibody or antibody fragmentspecifically binds to a lectin complement component, such as MASP-1,MASP-2, MASP-3, Map19, Map44, C4, C4a, C4b, C2, C2a and C2b. Theantibody is a monoclonal antibody. The antibody fragment is a Fabfragment, a Fab′ fragment, a F(ab′)2 fragment, or an ScFv fragment. Theantibody is a chimeric antibody. The antibody or antibody fragment ishumanized. Preferably, the antibody or fragment thereof is soluble andbinds to or inhibits a component or factor of the human alternativecomplement pathway.

Examples of agents that inhibit or reduce the activity of the complementpathway include, but are not limited to, cinryze, berinert, rhucin,eculizumab, pexelizumab, ofatumumab, TNX-234, compstatin/POT-4, PMX-53,rhMBL, human CD55, BCX-1470, C1-INH, SCR1/TP10, CAB-2/MLN-2222,mirococept, sCR1-sLe⁷/TP-20, TNX-558, TA106, Neutrazumab,anti-properdin, HuMax-CD38, ARC1905, JPE-1375, and JSM-7717.

Examples of agents that inhibit or reduce the activity of the lectinpathway include, but are not limited to, C1-inh, antithrombin, sunflowerMASP inhibitors SFMI-1 or SFMI-2, or SMGI inhibitors SGMI-1 or SGMI-2.Some preferred agents that inhibit the complement pathway are disclosedin US Publication 2013/0149373, WO 2013/093762, WO 2010/136311, WO2009/056631, which are hereby incorporated by reference in theirentireties. Other agents are disclosed in Wagner, E. et al., Nature Rev,2010, 9:43-56, Mucke, H. Et al., IDrugs, 2010, 13(1): 30-37, Ma, K. N.et al., Invest Ophthalmol Vis Sci, 2010, 51(12): 6776-6783, and RicklinD. et al, Nat Biotech, 2007, 25(11):1265-1275, which are herebyincorporated by reference in their entireties.

The compositions and methods described herein are also useful forinhibiting or reducing photoreceptor cell death by inhibiting orreducing complement pathway activity. Photoreceptor cells, together withphotosensitive retinal ganglion cells (RGCs) are responsible forconverting light into electric signals. Photoreceptor cell degenerationplays a significant role in retinal detachment and subsequent loss ofvision. Thus, prevention or reduction of photoreceptor cell deathalleviates or prevents permanent or significant vision loss. Untreatedretinal detachment or sustained photoreceptor cell death causes loss anddeath of retinal ganglion cells.

Photoreceptor cells are primarily distinguished from other retinal cellsby their morphology. Photoreceptor cells are divided into rod and conecells, as defined by their morphology and functional photopigments used.They are localized in the outer part of the retina; their nuclei arefound in the outer nuclear layer (ONL) while their-outer segments areoriented toward the retinal pigment epithelium (RPE). Structurally, conecells are somewhat shorter than rods, but wider and tapered, having acone-like shape at their outer segment where a pigment filters incominglight. Rod cells are longer than cone cells and leaner, and the pigmenton the outer side toward the RPE. Unique extracellular markers have alsobeen identified for photoreceptors. Examples of such markers areCacna2d4, Kcnv2, Pcdh21, recoverin, Rho4D2, and transducin. Antibodiesthat specifically bind to a photoreceptor-specific marker can be used toidentify photoreceptors.

Photoreceptor cell death can be measured by methods known in the art.For example, immunohistochemical analysis can be used by staining withapoptotic or cell death markers known in the art. The examples describedherein demonstrate TUNEL-staining of retinal cross-sections to identifycell death, and correlated to cell morphology to identifyphotoreceptor-specific cell death.

Spectral domain optical coherence tomography (SD-OCT) is also used fordetailed and non-invasive evaluation of the retinal architecture invivo. SD-OCT accurately reflects retinal morphological changes thatoccur during retinal disease progression, including retinal detachment.

Alternatively, photoreceptor cell death can be measured by assessingphotoreceptor cell function. Loss in photoreceptor cell functionindicates loss or death of photoreceptor cells. Electroretinography(ERG) analysis is a method known in the art for assessing photoreceptorfunction and neural responses. Advantages of this technique includenon-invasiveness, and objective evaluation of retinal function on alayer-by-layer basis. In brief, the flash ERG is assessed in a darkadapted eye. The initial a-wave (initial negative deflection) isprimarily derived from photoreceptors where the second half of thea-wave is a combination of photoreceptors, bipolar, amacrine, and mullercells. The b-wave (positive deflection) originates in retinal cells thatare post-synaptic to the photoreceptors and are used as a readout forphotoreceptor function.

Other methods for measuring photoreceptor function include standard eyeexaminations that are known in the art. For example, a Snellen chart, orvariations thereof, containing letters and/or numbers is used todetermine visual acuity. Decreased ability to distinguish and recognizethe letters and/or numbers indicates loss of photoreceptor function orvisual acuity. An increased ability to distinguish and recognize theletters and/or numbers after treatment indicates efficaciousness of thetreatment.

The compositions and methods described herein are useful for thepreservation of vision can be measured by methods known in the art. Forexample, preservation of vision can be measured by assessing retinalcell activity (i.e., photoreceptor cell activity) byelectroretinography, visual acuity by eye chart tests, and peripheralvision by visual field tests, as described herein.

The subject is a mammal in need of such treatment, e.g., a subject thathas been diagnosed with an ocular disorder associated withcomplement-mediated retinal cell death. For example, the subject hasbeen diagnosed with retinal detachment or a predisposition thereto orhas been identified as having experienced a head injury such astraumatic brain injury (TBI). The mammal is, e.g., a human, a primate, amouse, a rat, a dog, a cat, a horse, as well as livestock or animalsgrown for food consumption, e.g., cattle, sheep, pigs, chickens, andgoats. For example, the mammal is a performance mammal, such as aracehorse or racedog (e.g., greyhound). Preferably, the mammal is ahuman. In preferred embodiments, the subject does not comprisegeographic atrophy (GA), e.g., the subject has not been diagnosed withgeographic atrophy of the eye.

Subjects at risk for retinal detachment are individuals that have, forexample, nearsightedness (or severe myopia), previous cataract surgery,severe trauma, previous retinal detachment in either eye, family historyof retinal detachment, macular degeneration (i.e., wet maculardegeneration), diabetic retinopathy (i.e., proliferative diabeticretinopathy), retinopathy of prematurity, eclampsia, homocysteinuria,malignant hypertension, inflammatory conditions (i.e., uveitis orscleritis), glaucoma, retinoblastoma, metastatic cancer that spreads tothe eye, choroidal melanoma, haemangioma, Stickler syndrome, VonHippel-Lindau disease, AIDS, and smoking. Other subjects at risk forretinal detachment are individuals that engage in activities withincreased risk for trauma or injury to or in the proximity of the eye.Examples of such subjects include, but are not limited to, boxers,wrestlers, military personnel, young males.

A subject that is suffering from retinal detachment is identified bydiagnosis by a physician or clinician using methods known in the art, orsubjects that are presenting any symptoms of retinal detachment.Symptoms of retinal detachment include, but are not limited to, presenceof floaters in the field of vision, presence of flashes of light,increases in number or frequency of floaters or flashes of light, shadowor curtain over partial field of vision, photoreceptor cell death, lossin peripheral vision, central vision loss, decrease in visual acuity,and blindness. Conversely, GA is a sharply demarcated or completeatrophy of the retinal pigmented epithelium that may or may not involvethe fovea. Loss of the RPE results in progressive visual decline.

The compositions described herein are administered topically,intraocularly, intravitreally, subretinally, or systemically.Intraocular administration is preferred. Also preferred are intravitrealand systemic administration. Preferably, the compositions describedherein are administered by intraocular injection or systemically. In apreferred embodiment, the composition is administered shortly afterdiagnosis of retinal detachment, or appearance of a symptom of retinaldetachment. In some aspects, the composition is administered within 1minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22hours, or 24 hours, 30 hours, 36 hours, 42, hours, 48 hours, 56 hours or72 hours after retinal detachment or appearance of a symptom of retinaldetachment. Because of the location of the cell death, the inhibitorsare preferably delivered subretinally, e.g., using a fine needle fortreatment and reduction of cell death associated with retinaldetachment.

The composition further comprises a pharmaceutically acceptable carrierand/or ophthalmic excipient. Exemplary pharmaceutically acceptablecarrier include a compound selected from the group consisting of aphysiological acceptable salt, poloxamer analogs with carbopol,carbopol/hydroxypropyl methyl cellulose (HPMC), carbopol-methylcellulose, carboxymethylcellulose (CMC), hyaluronic acid, cyclodextrin,and petroleum.

All compounds of the invention are purified and/or isolated.Specifically, as used herein, an “isolated” or “purified” smallmolecule, nucleic acid molecule, polynucleotide, polypeptide, orprotein, is substantially free of other cellular material, or culturemedium when produced by recombinant techniques, or chemical precursorsor other chemicals when chemically synthesized. Purified compounds areat least 60% by weight (dry weight) the compound of interest.Preferably, the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by weight the compound ofinterest. For example, a purified compound is one that is at least 90%,91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compoundby weight. Purity is measured by any appropriate standard method, forexample, by column chromatography, thin layer chromatography, orhigh-performance liquid chromatography (HPLC) analysis. A purified orisolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid(DNA)) is free of the genes or sequences that flank it in its naturallyoccurring state. Purified also defines a degree of sterility that issafe for administration to a human subject, e.g., lacking infectious ortoxic agents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybridgene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones. Isolatednucleic acid molecules also include messenger ribonucleic acid (mRNA)molecules.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid and the phrase “nucleic acid sequence” refers tothe linear list of nucleotides of the nucleic acid molecule, the twophrases can be used interchangeably.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component, alone or in a combination, to providethe desired effect. For example, by “an effective amount” is meant anamount of a compound, alone or in a combination, required to prevent PVRin a mammal. Ultimately, the attending physician or veterinarian decidesthe appropriate amount and dosage regimen.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. The terms “preventing” and“prevention” refer to the administration of an agent or composition to aclinically asymptomatic individual who is susceptible or predisposed toa particular adverse condition, disorder, or disease, and thus relatesto the prevention of the occurrence of symptoms and/or their underlyingcause.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below.

All published foreign patents and patent applications cited herein areincorporated herein by reference. Genbank and NCBI submissions indicatedby accession number cited herein are incorporated herein by reference.All other published references, documents, manuscripts and scientificliterature cited herein are incorporated herein by reference. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E are images and graphs demonstrating the activity of thealternative complement pathway during retinal detachment. FIG. 1A is abar graph showing results from an ELISA of Fb using human vitreous frompatients with retinal detachment compared to macular hole asnon-detached control samples (control n=4, retinal detachment n=9). FIG.1B is an image of the mouse model of retinal detachment. The dotted lineoutlines the region of retina that is detached, approximately 60%. FIG.1C is a line graph showing a time course for gene expression of Fb andCd55 in the retina of mice following RD. The line connecting the closedcircles tracks Fb expression and the line connecting the closed squarestracks Cd55 expression at intervals over a period of 48 hours. FIG. 1Dis a bar graph showing a time course for Fb protein following retinaldetachment in mice. FIG. 1E is a bar graph showing gene expression ofCd55 in the ONL 24 hours after RD. (Fb=Factor b, RD=retinal detachment,ONL=outer nuclear layer) (ns=not significant, *≦0.05, **≦0.01,***≦0.001).

FIGS. 2A-F are graphs and images demonstrating apoptosis in complementdeficient mice after retinal detachment. FIG. 2A is an image ofrepresentative TUNEL labeling in C3−/− mice and wild type control(C57BI6) mice 24 hours after retinal detachment (scale bar=50 μm). FIG.2B is a bar graph depicting quantitation of TUNEL cells in the ONL ofC3−/− mice and wild type control. FIG. 2C is an image of representativeTUNEL labeling in the ONL of mice injected with an antibody against C3compared to IgG control. FIG. 2D is a bar graph depicting quantitationof TUNEL cells in the ONL of mice injected with an antibody against C3compared to IgG control. FIG. 2E is an image showing representativeTUNEL labeling in the ONL of C3−/− mice injected with PBS, control, orCVF (to activate the complement system). FIG. 2F is a bar graphdepicting quantitation of TUNEL positive cells in the ONL of C3−/− miceinjected with PBS, control, or CVF (to activate complement in C3−/−mice). (WT=wild type, ONL=outer nuclear layer, CVF=cobra venom factor)(**≦0.01, ****≦0.0001, scale bar=50 μm).

FIGS. 3A-D are graphs and images demonstrating apoptosis in alternativepathway deficient mice after retinal detachment. FIG. 3A is an image ofrepresentative TUNEL labeling 24 hours after retinal detachment in Fb−/−mice and wild type control. FIG. 3B is bar graph depicting quantitationof TUNEL positive cells from Fb−/− mice and wild type control 24 hoursafter RD. FIG. 3C is an image of representative TUNEL labeling 24 hoursafter RD in mice injected with a Fd neutralizing antibody and IgGisotype control. FIG. 3D is a bar graph depicting quantitation of TUNELpositive cells from mice injected with a Fd neutralizing antibody andIgG isotype control 24 hours after RD (WT=wild type, Fb−/−=Factor bknock out mice, anti Fd ab=antibody against Factor d, RD=retinaldetachment, ONL=outer nuclear layer) (****≦0.0001, scale bar=50 μm).

FIGS. 4A-E are graphs and images demonstrating the role of hypoxia inthe retina following retinal detachment. FIG. 4A is an image ofrepresentative IHC of the ONL labeled with hypoxyprobe (brown DAB)comparing the detached portion of the retina (right panel) to theattached retina (left panel) in the same retina 24 hours after RD. FIG.4B is a bar graph of in vivo oxygen concentrations taken in the retina24 hours after RD in an attached retina (right eye) compared to thedetached retina (left eye). FIG. 4C is an image of representative TUNELlabeling in the ONL 24 hours after RD in mice kept in room air (leftpanel) compared to mice kept in 75% oxygen (right panel). FIG. 4D is abar graph depicting quantitation of TUNEL positive cells in mice kept inroom air for 24 hours after RD compared to mice kept in 75% oxygen. FIG.4E is a bar graph showing RTPCR for gene expression of Fb in the retina24 hours after RD comparing mice kept in room air compared to mice keptin 75% oxygen. (RD=retinal detachment, ONL=outer nuclear layer) (ns=notsignificant, *≦0.05, ****≦0.0001, scale bar=50 μm).

FIG. 5 is a table of human patient data for ELISA samples. Summary ofrelevant patient history of the samples collected for ELISA ofalternative pathway proteins. An OCT of the retinal detachment forpatient NR-13, indicated in bold, is shown in FIGS. 6A.

FIGS. 6A-E are graphs and images depicting data from human samples ofretinal detachment. FIG. 6A is an image of retinal detachment observedin patient NR13 of FIG. 5. The area outlined by the dotted line is thearea of detachment and the solid line the plane of the OCT image shownin FIG. 6B. FIG. 6B is an OCT image taken through the detached area atthe plane of the detachment marked by the solid line in FIG. 6A. FIG. 6Cis a bar graph of results from an ELISA of complement Factor d. FIG. 6Dis a bar graph of results from an ELISA of complement C5. FIG. 6E is abar graph of results from an ELISA of C3 in the vitreous of patientswith a detached retina compared to macular hole, as a non-detachedcontrol. (ns=not significant).

FIGS. 7A-D are images and a graph depicting a mouse model of retinaldetachment. FIG. 7A is a picture illustrating the typical retinaldetachment observed in mice after an injection of Provisc® into thesub-retinal space. The dotted white line is outlining the region of theretina that is detached. The dotted black line indicates the crosssectional region in FIG. 7B. FIG. 7B is an image of a cross section ofthe eye (dotted red line in FIG. 7A) showing the detached retina. Thedotted lines outline the detached portion of the retina. The boxesindicate the regions shown in FIG. 7C. FIG. 7C is a bar graph showing atime course of TUNEL labeling in the ONL after retinal detachment. FIG.7D is an image of TUNEL labeling following a time course after retinaldetachment. The peak of apoptosis is at 24 hours. (ONL=outer nuclearlayer, scale bar=50 μm).

FIG. 8 is a graph demonstrating activation of the lectin and classicalcomplement pathways in the mouse RD model (A) RTPCR showing a timecourse for Masp2 (lectin pathway) and C1q (classical pathway) geneexpression after retinal detachment. (ns=not significant, *≦0.05,**≦0.01).

FIGS. 9A and B are images showing laser capture micro-dissection of theONL. FIG. 9A is an image of a cross section of the retina including thedetached space stained with toluidine blue dye. FIG. 9B is an image ofthe same cross section shown in FIG. 9A after cutting out the ONL usinglaser capture micro-dissection, outlined in a dotted line. (ONL=outernuclear layer, INL=inner nuclear layer, GCL=ganglion cell layer).

FIGS. 10A-D are images and graphs demonstrating the role of the lectinand classical complement pathways in ONL cell death after retinaldetachment. FIG. 10A is an image of representative TUNEL labeling 24hours after RD in Mbl−/− and wild type, control, mice. FIG. 10B is a bargraph depicting quantitation of TUNEL positive nuclei in the ONL 24hours after RD in MbI−/− and wild type control mice. FIG. 10C is animage of representative TUNEL labeling 24 hours after RD in C1q−/− andwild type, control, mice. FIG. 10D is a bar graph depicting quantitationof TUNEL positive nuclei in the ONL 24 hours after RD in C1q−/− and wildtype control mice. (RD=retinal detachment, ONL=outer nuclear layer)(ns=not significant, *≦0.05, **≦0.01, scale bars=50 μm).

FIG. 11 is a schematic depicting the three complement pathways.

DETAILED DESCRIPTION

The retina sends visual images to the brain through the optic nerve.Retinal detachment is a disorder of the eye in which the retina peelsaway from its underlying layer of support tissue. Detachment of theretina causes vision loss and blindness, and if left untreated, thevision loss or blindness can be permanent.

Photoreceptor cell death occurs when the outer segments are physicallyseparated from the underlying retinal pigment epithelium (RPE) andchoroidal vasculature (Zacks, D. N., et al, Invest Ophthalmol Vis Sci,2006, 47:1691-1695). While numerous pathological changes occur in thedetached retina, studies in human patient samples and in animal modelshave shown that photoreceptor cell death is induced as early as 12 hoursand peaks at around 2-3 days after RD (Yu, J. et al, Invest Ophalmol VisSci, 2012, 53:8146-8153). However, the underlying processes thatfacilitate this death have remained elusive. Results described hereindemonstrate that photoreceptor degeneration correlates with a rise oramplification of the complement system. Without wishing to be bound bytheory, the complement system mounts an immune response against thephotoreceptors in the damaged retina to specifically target these cellsfor removal.

The complement system is an intricate innate immune surveillance pathwaythat is able to discriminate between healthy host tissue, diseased hosttissue, apoptotic cells and foreign invaders while modulating theelimination and repair of host tissue accordingly (Yanai, R. et al., AdvExp Med Biol, 2012, 946:161-183). Consisting of serum and tissueproteins, membrane-bound receptors, and a number of regulatory proteins,the complement system is a hub-like network that is tightly connected toother systems; it comprises three key pathways: the classical, lectinand alternative pathways (Yanai, R. et al., Adv Exp Med Biol, 2012,946:161-183; and Ricklin, D. et al, Nat Immunol, 2010, 11:785-797).Within the ocular microenvironment the alternative complement pathwayexhibits low levels of constitutive activation to ensure theintermittent probing of host self cells, which express inhibitors ofcomplement for protection from activation. On the other hand, damaged ordiseased host cells down-regulate membrane-bound inhibitors ofcomplement (CD55), allowing for targeted clearance.

The data described herein demonstrate the surprising results thatinhibiting or reducing the alternative complement pathway activityprevent photoreceptor cell death after retinal detachment, therebyresulting in preserving vision. Exemplary agents such as those describedin Table 1 below are useful to improve the pathological aspects andsymptoms of RD.

TABLE 1 Route of Phase of adminis- clinical DRUG Mechanism tration trialCOMPANY FCFD4514S Anti-factor Intravitreal 2^(3,4,6) Genentech Dantibody POT-4 C3 inhibitor Intravitreal 2⁵ Alcon (Compstatin) ARC1905Aptamer- Intravitreal 1^(1,2) Ophthotec based completed C5 inhEculizumab Anti-C5 oral 3 Alexion. (Soliris) antibody LFG316^(7,8)Anti-C5 Intravitreal Novartis antibody TA-106 Complement Taligen factorTherapeutics, B (CFB) Alexion inhibitor Pharmaceuticals Anti-factorGenentech B antibody ¹Opthotech. ClinicalTrials.gov [Internet]. Bethesda(MD): National Library of Medicine (US). 2000 [Cited 2014 Oct. 7].Available from http://clinicaltrials.gov/show/NCT00950638 NLMIdentifier: NCT00950638. ²Opthotech. ClinicalTrials.gov [Internet].Bethesda (MD): National Library of Medicine (US). 2000 [Cited 2014 Oct.7]. Available from http://clinicaltrials.gov/show/NCT00709527 NLMIdentifier: NCT00709527. ³Genentech. ClinicalTrials.gov [Internet].Bethesda (MD): National Library of Medicine (US). 2000 [Cited 2014 Oct.7]. Available from http://clinicaltrials.gov/show/NCT01229215 NLMIdentifier: NCT01229215. ⁴Genentech. ClinicalTrials.gov [Internet].Bethesda (MD): National Library of Medicine (US). 2000 [Cited 2014 Oct.7]. Available from http://clinicaltrials.gov/show/NCT00973011 NLMIdentifier: NCT00973011 ⁵Potentia Pharmaceuticals, Inc.ClinicalTrials.gov [Internet]. Bethesda (MD): National Library ofMedicine (US). 2000 [Cited 2014 Oct. 7]. Available fromhttp://clinicaltrials.gov/show/NCT00473928 NLM Identifier: NCT00473928.⁶Genentech. ClinicalTrials.gov [Internet]. Bethesda (MD): NationalLibrary of Medicine (US). 2000 [Cited 2014 Oct. 7]. Available fromhttp://clinicaltrials.gov/show/NCT01602120 NLM Identifier: NCT01602120.⁷M. Roguska, et al. Generation and Characterization of LFG316, aFully-Human Anti-C5 Antibody for the Treatment of Age-Related MacularDegeneration [abstract]. In: ARVO 2014 Annual Meeting Abstracts. ARVOAnnual Meeting. 2014 May 4-8; Orlando, FL. Abstract No. 3432 - C0281.⁸A. Carrion, et al. Characterization of the Stoichiometry of HumanComplement C5 Binding to LFG316 [abstract]. In: ARVO 2014 Annual MeetingAbstracts. ARVO Annual Meeting. 2014 May 4-8; Orlando, FL. Abstract No.3432-C0280.

Several of the therapies described in the above table are antibodies. Toscreen for antibodies which bind to a particular epitope on the antigenof interest, a routine cross-blocking assay such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. Alternatively, epitopemapping, e.g. as described in Champe et al. (1995) J. Biol. Chem.270:1388-1394, can be performed to determine whether the antibody bindsan epitope of interest, as described in WO2009061910A1 (incorporated inits entirety by reference herein). Example antibodies from Table 1 aboveare described briefly below.

Anti-Factor B Antibodies

Anti-factor B antibodies are selected using a factor B antigen derivedfrom a mammalian species. Preferably the antigen is human factor B.However, factor Bs from other species such as murine factor B can alsobe used as the target antigen. The factor B antigens from variousmammalian species may be isolated from natural sources. In otherembodiments, the antigen is produced recombinantly or made using othersynthetic methods known in the art. The antibody selected will normallyhave a sufficiently strong binding affinity for the factor B antigen.For example, the antibody may bind human factor B with a Kd value of nomore than about 5 nM, preferably no more than about 2 nM, and morepreferably no more than about 500 pM.

Antibody affinities may be determined by a surface plasmon resonancebased assay (such as the BiAcore assay as described in Examples);enzyme-linked immunoabsorbent assay (ELISA); and competition assays(e.g. RIA's), for example. Also, the antibody may be subject to otherbiological activity assays, e.g., in order to evaluate its effectivenessas a therapeutic. Such assays are known in the art and depend on thetarget antigen and intended use for the antibody. Examples include theHUVEC inhibition assay (as described in the Examples below); tumor cellgrowth inhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and in vitro and invivo assays described below for identifying factor B antagonists.

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest. In a preferredembodiment, the anti-factor 13 antibodies are selected using a uniquephage display approach. The approach involves generation of syntheticantibody phage libraries based on

single framework template, design of sufficient diversities withinvariable domains, display of polypeptides having the diversifiedvariable domains, selection of candidate antibodies with high affinityto target factor B antigen, and isolation of the selected antibodies.

Examples of anti-factor B antibodies are described in WO2009061910A1,WO2013177035, WO2008140653, and US 20050260198 (each of which is herebyincorporated by reference).

Anti-Factor Bb

An exemplary antibodies specific for factor B for use in inhibiting theactivity of C3bBb or PC3bBb complexes, such an antibody inhibits theproteolytic activity of factor B in C3/C5 convertases. Anti-Factor Fbantibodies selectively block the binding of factor Bb to the PC3bBcomplex without inhibiting classical pathway activation as described inWO2013152020 (incorporated by reference in its entirety herein).

Anti-Factor D

Further example therapies comprise antibodies that target factor D. Anexample anti-Factor D antibody comprises light chain HVR-1 comprisingITSTDIDDDMN (SEQ ID NO: 1), light chain HVR-2 comprising GGNTLRP (SEQ IDNO: 2), and light chain HVR-3 comprising LQSDSLPYT (SEQ ID NO: 3) andmay comprise amino acid substitutions provided that they preserve itsantibody binding properties, e.g., as described in US 20140065137,incorporated by reference in its entirety herein. An additionalexemplary factor D antibody or a binding fragment thereof may bind tothe same epitope on human Factor D as monoclonal antibody 166-32 and isproduced by the hybridoma cell line deposited under ATCC AccessionNumber HB-12476 as described in U.S. Pat. No. 8,124,090, incorporated inits entirety by reference herein. Another exemplary anti-factor Dantibody is U.S. Pat. No. 8,372,403 (incorporated in its entirety byreference herein).

Anti-C3 Antibody

An example anti C3 antibody selectively blocks the binding of factor Bto C3b without inhibiting the classical pathway activation. These typesof antibodies, as described in WO2013152024 do not inhibit theinteraction of C3b to C5 and therefore have a unique function ininhibiting the alternative pathway. Alternative anti-C3 antibodies caninhibit complement activation by way of inhibition of C3b function, asdescribed in US 20100111946. Each of the references are hereinincorporated by reference in their entireties.

Anti-Properdin Antibody

An exemplary anti-Properdin antibody is that described in WO 2013006449(incorporated in its entirety by reference herein) with the epitopecomprising amino acids of the sequence: RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP(SEQ ID NO: 4).

Anti-C5 Antibody

The prevention of C5a generation with antibodies during the arrival ofsepsis in rodents has been shown to greatly improve survival, whilerelated findings were made when the C5a receptor (C5aR) was blocked,using either antibodies or a small molecular inhibitor (Landes, U., etal., Anti-c5a ameliorates coagulation/fibrinolytic protein changes in arat model of sepsis. American Journal Of Pathology, 2002, 160(5): p.1867; Riedemann, N. C., R, F. Guo, and P. A. Ward, A key role ofC5a/C5aR activation for the development of sepsis. Journal of LeukocyteBiology, 2003. 74(6): p. 966). An additional exemplary anti-C5 antibodyis described in WO1995029697. An additional example antibody is themonoclonal antibody designated MAb137-26, which binds to a sharedepitope of human C5 and C5a, as described in U.S. Pat. No. 8,372,404.Each of the references are herein incorporated by reference in theirentireties.

Non antibody inhibitors are also listed in Table 1 above, briefdescriptions of these inhibitors are found below.

POT-4

POT-4 is a derivative of the cyclic peptide Compostatin. It is capableof binding to human complement factor C3 (Potentia pharmaceuticals,http://www.potentiapharma.com/products/pot4.htm). POT 4 suppressescomplement activation by preventing the formation of key elements withinthe proteolytic cascade, thus impeding local inflammation, upregulationof angiogenic factors and subsequent tissue damage (O. S. Punjabi and P.K. Kaiser, Review of Ophthalmology. Oct. 4, 2012.http://www.reviewofophthalmology.com/content/d/retinal_insider/c/36952).

ARC 1905

ARC 1905 is a PEGylated, stabilized aptamer targeting complement factorC5, blocking the cleavage of C5 into C5a and C5b fragments (ARC1905inhibits C5—Dry/Wet AMD Intravitreal,http://www.amdbook.org/content/arc1905-inhibits-c5-drywet-amd-intravitreal).Like POT-4, it is similarly selective for a centrally positionedcomponent within the cascade, although exerting its effect furtherdownstream (0. S. Punjabi and P. K. Kaiser, Review of Opthomology. Oct.4, 2012.http://www.reviewofophthalmology.com/content/d/retinal_insider/c/36952).

TA-106

TA-106 inhibits Factor B, a serine proteinase that is unique to thealternative pathway and exists upstream of complement proteins targetedby many other drugs, including complement 3 (C3) and C5. (BioCentury,The Bernstein Report on BioBusiness. Aug. 13, 2007). It is primarilybeing investigated as an inhaled formulation in the treatment of severe,chronic asthma refractory to current therapies and is recently beingstudied for macular degeneration (0. S. Punjabi and P. K. Kaiser, Reviewof Ophthalmology. Oct. 4, 2012.http://www.reviewofophthalmology.com/content/d/retinal_insider/c/36952).

Many of the therapies described above were developed for the treatmentof Age-related Macular Dystrophy (AMD). However, data described hereindemonstrates the applicability of inhibitors of the alternativecomplement pathway in the treatment of retinal detachment.

The methods and compositions described herein provide a non-surgicaltherapy for retinal detachment.

Retinal Detachment

There are three types of retinal detachment: rhegmatogenous, exudative,and tractional. Rhegmatogenous retinal detachment occurs due to a breakin the retina that allows fluid to pass from the vitreous space into thesubretinal space between the sensory retina and the retinal pigmentepithelium. Chronic retinal atrophy or a result of vitreous traction cancause retinal breaks. Retinal breaks include retinal holes, retinaltears, and retinal dialyses. Exudative retinal detachment (also known asserous or secondary retinal detachment) occurs due to inflammation,injury or vascular abnormalities that result in fluid accumulatingunderneath the retina without the presence of a hole, tear, or break. Insome rare cases, exudative retinal detachment can be caused by thegrowth of a tumor on the layers of tissue beneath the retina (i.e.,choroidal melanoma). Tractional retinal detachment occurs when fibrousor fibrovascular tissue pulls the sensory retina from the retinalpigment epithelium, often caused by injury, inflammation, orneovascularization.

The compositions and methods described herein are particularly usefulfor treating a subject that has suffered from a trauma-induced retinaldetachment. Trauma-induced retinal detachment, as used herein, refers toa retinal detachment resulting from a physical trauma or injury, forexample, blunt or penetrating blows to the eye or areas surrounding theeye, concussion to the head, or previous eye surgery. Trauma-inducedretinal detachment can occur in high impact sports (i.e., boxing,karate, kickboxing, American football, high intensity weightlifting), inhigh speed sports (i.e., automobile racing, sledding), or in activitiesthat increase pressure on or within the eye itself, or include rapidacceleration and deceleration. Active military personnel are also atincreased risk for retinal detachment, for example, as a result ofinjuries sustained from improvised explosive devices (IEDs) blasts inthe field.

Symptoms of retinal detachment include presence of flashes of light(photopsia), floaters, or a shadow or curtain in the field of vision.The flashes of light may be sudden, and very brief in the extremeperipheral part of the vision. Floaters may look like small debris,spots, hairs, or string that float in the field of vision. The shadow orcurtain may appear in a portion of the field of vision, and develops orspreads as the detachment progresses. Other symptoms include sudden ordramatic increase in the number of floaters or flashes of light. Othersymptoms include a slight feeling of heaviness in the eye, centralvisual loss, decrease in visual acuity, any vision loss, or blindness.

Retinal detachment can occur at any age, but it is more common inmidlife and later. Conditions that can increase the chance of a retinaldetachment include, for example, nearsightedness (or severe myopia),previous cataract surgery, severe trauma, previous retinal detachment ineither eye, family history of retinal detachment, proliferative diabeticretinopathy, retinopathy of prematurity, eclampsia, homocysteinuria,malignant hypertension, inflammatory conditions (i.e., uveitis orscleritis), glaucoma, retinoblastoma, metastatic cancer that spreads tothe eye, choroidal melanoma, haemangioma, Stickler syndrome, VonHippel-Lindau disease, AIDS, and smoking.

Diagnosis of retinal detachment is performed using fundus photography,ophthalmography, or medical ultrasonography.

Generally, current treatment methods for treating retinal detachment aimto find and seal all retinal breaks, and relieve present and futurevitreoretinal traction. These treatment procedures include, but are notlimited to, cryopexy (freezing) and laser photocoagulation, scleralbuckle surgery, pneumatic retinopexy, and vitrectomy.

The compositions and methods described herein provide the advantage overthe current treatment methods by being non-surgical, and having lessassociated complications. The compositions and methods disclosed hereinprevent photoreceptor cell death, thereby allowing the retinal toreattach post-injury. Furthermore, administration of the compositionsdisclosed herein is useful for preventing progression of retinaldetachment when delivered shortly after diagnosis of retinal detachmentor appearance of a symptom of retinal detachment. For example, thecomposition is administered within 6 hours, 12 hours, 18 hours, 24hours, 30 hours, 36 hours, 42, hours, 48 hours, 56 hours or 72 hoursafter retinal detachment or appearance of a symptom of retinaldetachment. Methods described herein can also be performed incombination with any of the current treatment methods or surgicalprocedures used to treat retinal detachment or reattach the detachedretina.

Complement Pathway

The complement system is comprised of various soluble and surface-boundcomplement components, receptor and regulators that are initiated byinteraction of several pattern-recognition receptors with foreignsurface structures. Depending on the activation trigger, the complementcascade follows one of three pathways: the classical, lectin oralternative pathway. The three complement pathways follow a differentsequence of “early” cleavage reactions that all lead to the assembly ofa protease named C3 convertase. The C3 convertase then cleaves proteinC3 into C3a and C3b, which in turn causes a cascade of signaling andcleavage of other complement components, eventually initiating theformation of the membrane attack complex (MAC). MAC comprises C5b, C6,C7, C8, and polymeric C9, forming a transmembrane channel that causesosmotic lysis of the target cell. A schematic of the pathways and theircomponents is shown in FIG. 11.

The alternative pathway is initiated by the hydrolysis of circulating C3to expose an internal thioester group. This phenomenon is referred to as“C3 tickover”. The hydrolysed C3 then binds alternative-pathway-specificproteins factor B, factor D, and properdin, to form activatedC3-convertase. At this point, the alternative complement pathwayconverges with the classical and lectin pathways in the cleavage of C3into active fragments of C3a and C3b by the C3 convertases. In anamplification loop, factor B can also bind C3b and is processed byfactor D to generate more C3 convertase, thereby initiating anacceleration of C3b production. C3b initiates the formation of the twoC5 convertases, which cleaves C5 to C5a and C5b. C5b initiates theassembly of the membrane attack complex (MAC), a pore that is formed bycomponents C5b, C6, C7, C8, and multiple units of C9 (C5b-9), andultimately leads to cell lysis and death.

The lectin pathway is activated by binding of mannose-binding lectin(MBL) to mannose residues on the pathogen surface, which activates theMBL-associated serine proteases, MASP-1, and MASP-2 (very similar to C1rand C1s, respectively), which can then split C4 into C4a and C4b and C2into C2a and C2b. C4b and C2b then bind together to form C3-convertase.RT-PCR analysis of components of the lectin pathway in models of retinaldetachment show that the lectin complement pathway is also highlyregulated in retinal detachment.

The methods and compositions disclosed herein inhibit or reduce theactivity of a complement pathway. Preferably, the complement pathway isthe alternative complement pathway. Inhibiting or reducing the activityof a complement pathway, as used herein, refers to the modulation thetranscription stability, translation, modification, localization,cleavage, or function of a polynucleotide or polypeptide encoding anyone of the selected from properdin (Factor p), factor B, factor Ba,factor Bb, factor D, C2, C2a, C3a, C5, C5a, C6, C7, C8, C9, and C5b-9.Preferably, the modulation results in an inhibition or decrease in theactivity (i.e., function) or expression of the complement component.

In some embodiments, the agent that inhibits or reduces complementpathway activity comprises an antibody or an antibody fragment.Preferably, the antibody specifically binds to the properdin (Factor p),factor B, factor Ba, factor Bb, factor D, C2, C2a, C3a, C3b, C5, C5a,C5b, C6, C7, C8, C9, and C5b-9. In a preferred embodiment, the antibodyinhibits the activity (function) of the complement component, forexample, preventing or reducing the binding to the cognate receptor, thebinding of the receptor to the ligand, cleavage, or activation. Examplesof therapeutic antibodies include eculizumab, pexelizumab, ofatumumab,TNX-234, TNX-558, TA106, neutrazumab, and anti-properdin. An exemplaryantibody specifically binds to factor B (Fb).

The term “antibody” as used herein includes whole antibodies and anyantigen binding fragment (i. e., “antigen-binding portion”) or singlechains thereof. A naturally occurring “antibody” is a glycoproteincomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains. CH1, CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRsarranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Reichmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

Human monoclonal antibodies can be prepared by using trioma technique;the human B-cell hybridoma technique (Kozbor, et al., 1983 Immunol Today4: 72); and the EBV hybridoma technique to produce human monoclonalantibodies (Cole, et al., 1985 In: Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies maybe utilized and may be produced by using human hybridomas (Cote, et al.,1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming humanB-cells with Epstein Barr Virus in vitro (Cole, et al., 1985 In:Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In addition, human antibodies can also be produced usingadditional techniques, including phage display libraries. (Hoogenboomand Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen (PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv (scFv) molecules.

Services are currently offered commercially by companies (i.e.,Immunomedics Inc., 300 The American Road, Morris Plains, N.J. 07950,USA; Antitope Ltd., Babraham Research Campus, Babraham, Cambridge CB223AT, United Kingdom; and GenScript USA Inc., 860 Centennial Ave.,Piscataway, N.J. 08854, USA) for the production of humanized antibodies.

The term “antigen binding portion” of an antibody, as used herein,refers to one or more fragments of an intact antibody that retain theability to specifically bind to a given antigen (e.g., C3b). Antigenbinding functions of an antibody can be performed by fragments of anintact antibody. Examples of binding fragments encompassed within theterm “antigen binding portion” of an antibody include a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; aF(ab)₂ fragment, a bivalent fragment comprising two Fab fragments linkedby a disulfide bridge at the hinge region; an Fd fragment consisting ofthe VH and CH1 domains; an Fv fragment consisting of the VL and VHdomains of a single arm of an antibody; a single domain antibody (dAb)fragment (Ward et al. 1989 Nature 341:544-546), which consists of a VHdomain or a VL domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by an artificial peptide linker that enables them to be made asa single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl.Acad. Sci. 65:5879-5883). Such single chain antibodies include one ormore “antigen binding portions” of an antibody. These antibody fragmentsare obtained using conventional techniques known to those of skill inthe art, and the fragments are screened for utility in the same manneras are intact antibodies. Antigen binding portions can also beincorporated into single domain antibodies, maxibodies, minibodies,interbodies, diabodies, triabodies, totrabodies. v-NAR and bis-scFv(see, e.g., Hollinger and Hudson. 2005. Nature Biotechnology, 23, 9,1126-1136).

Antigen binding portions can be incorporated into single chain moleculescomprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al., 1995 Protein Eng. 8(10): 1057-1062; andU.S. Pat. No. 5,641,870).

The term “binding specificity” or “specifically binds” as used hereinrefers to the ability of an individual antibody combining site to reactwith only one antigenic determinant, and therefore does not bind othercomplement components. The combining site of the antibody is located inthe Fab portion of the molecule and is constructed from thehypervariable regions of the heavy and light chains. Binding affinity ofan antibody is the strength of the reaction between a single antigenicdeterminant and a single combining site on the antibody. It is the sumof the attractive and repulsive forces operating between the antigenicdeterminant and the combining site of the antibody.

Other agents that inhibit or reduce complement pathway activity areserine protease inhibitors. The complement cascade relies upon theconsecutive cleavage and activation of several proteases. Proteases inthe complement cascade include, for example, C1r, C1s, C2a, MASP1,MASP2, factor D, and factor B. The protease inhibitor binds to theprotease and preferably prevents its cleavage function. Examples ofserine protease inhibitors are C1-Inh and rhucin.

Soluble complement regulators are also useful as agents that inhibit orreduce complement pathway activity. For example, the agent is a solubleform of a complement receptor that competes with endogenous complementreceptors, thereby reducing the complement pathway activity.Alternatively, the agent is a soluble form of an endogenous complementinhibitor that reduces complement pathway activity. For example, theagent is a soluble form of DAF/CD55 of CD59. Examples of solublecomplement regulators include sCR1/TP10, CAB-2/MLN-2222, mirococept, andsoluble CD55 mimetic.

Another agent that inhibits or reduces complement pathway activity is acomplement component inhibitor, such as a small molecule that interruptsprotein functions by steric hindrance or induction of conformationalchanges. The agent may be a peptide, a nucleotide, or a syntheticmolecule. For example, the agent may be an aptamer, which is asingle-stranded nucleotide that has molecular recognition propertiessimilar to those of antibodies but can be selected in an automatedhigh-throughput process known as SELEX. Aptamers that recognizecomplement components can provide complete blockage of downstreamcomplement activation. For example, anti-C5 aptamer (ARC1905) features asubnanomolar binding affinity for C5 and inhibits the cleavage of C5aand C5b. Other examples of complement component inhibitors includecompstatin/POT-4.

Anaphylatoxin receptor antagonists can also be used to inhibit or reducecomplement cascade signaling. Anaphylatoxins C3a and C5a are potentinflammatory mediators that binds to high affinity receptors. Theseantagonists are designed to bind to the receptor with high affinitywithout inducing any signaling activity, thereby inhibiting and reducingcomplement pathway activity. Examples of anaphylatoxin receptorantagonists include PMX-53, PMX-205, JPE-1375, and JSM-7717.

Nucleic acid expression vectors that encodes an anti-complement agentare also useful for inhibiting or reducing complement pathway activity.The anti-complement agent may be a polynucleotide or a polypeptide thatinhibits or reduces a complement component activity or expression. Thepolynucleotide may be an interfering RNA or an aptamer. The polypeptidemay be a complement inhibitor or receptor antagonist. For example, theagent is an AAV-expression vector comprising CD55.

Any of the agents described herein may be derivatized or modified usingmethods known in the art for modulating the pharmacological properties,such as stability, half-life, permeability, and affinity.

Pharmaceutical Compositions

For administration to a subject such as a human or other mammal (e.g.,companion, zoological or livestock animal), an agent that inhibits orreduces alternative complement pathway activity is desirably formulatedinto a pharmaceutical composition containing the active agent inadmixture with one or more pharmaceutically acceptable diluents,excipients or carriers. Examples of such suitable excipients for can befound in U.S. Publication 2009/0298785 (incorporated by reference hereinin its entirety), the Handbook of Pharmaceutical Excipients, 2nd Edition(1994), Wade and Weller, eds. Acceptable carriers or diluents fortherapeutic use are well-known in the pharmaceutical art, and aredescribed, for example, in Remington: The Science and Practice ofPharmacy, 20th Edition (2000) Alfonso R. Gennaro, ed., LippincottWilliams & Wilkins: Philadelphia, Pa. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical composition can contain as, or in addition to, thecarrier, excipient or diluent any buffering agent(s), suitablebinder(s), lubricant(s), suspending agent(s), coating agent(s),solubilizing agent(s), isotonifier(s), non-ionic detergent(s), and othermiscellaneous additives. Such additives must be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-disodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, there may be mentioned phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavoring agents can beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents can be also used, Preservativesmay be added to retard microbial growth, and may be added in amountsranging from 0.2%4% (w/v). Suitable preservatives for use with thepresent invention include phenol, benzyl alcohol, meta-cresol, methylparaben, propyl paraben, octadecyldimethylbenzyl ammonium chloride,benzalconium halides (e.g., chloride, bromide, iodide), hexamethoniumchloride, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, and 3-pentanol.

Isotonicifiers sometimes known as “stabilizers” may be added to ensureisotonicity of liquid compositions of the present invention and includepolyhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenyalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including such as inositol; polyethylene glycol; amino acid polymers:sulfur containing reducing agents, such as urea, glutathione, thiocticacid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodiumthio sulfate; low molecular weight polypeptides (i.e. <10 residues);proteins such as human serum albumin, bovine serum albumin, gelatin orimmunoglobulins; hydrophylic polymers, such as polyvinylpyrrolidonemonosaccharides, such as xylose, mannose, fructose, glucose;disaccharides such as lactose, maltose, sucrose and trisaccacharidessuch as raffinose; polysaccharides such as dextran. Stabilizers may bepresent in the range from 0.1 to 10,000 weights per part of weightactive protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation herein mayalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desirable to further provide an immunosuppressive agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The active ingredients may also beentrapped in microcapsule prepared, for example, by conservationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin micropheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions,Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, A. Osal, (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished, for example, by filtration through sterilefiltration membranes. Sustained-release preparations may be prepared.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theantibody variant, which matrices are in the form of shaped articles,e.g., films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions, Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol A. Ed. (1980),

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the anti-complement inhibitors, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the Lupron Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C. resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions,

A suitable formulation of the compositions disclosed herein is ahydrogel. A hydrogel is a colloidal gel formed as a dispersion in wateror other aqueous medium. Thus a hydrogel is formed upon formation of acolloid in which a dispersed phase (the polymer) has combined with acontinuous phase (i.e. water) to produce a viscous jellylike product;for example, coagulated silicic acid, A hydrogel is a three-dimensionalnetwork of hydrophilic polymer chains that are crosslinked througheither chemical or physical bonding. Because of the hydrophilic natureof the polymer chains, hydrogels absorb water and swell (unless theyhave already absorbed their maximum amount of water). The swellingprocess is the same as the dissolution of non-crosslinked hydrophilicpolymers. By definition, water constitutes at least 10% of the totalweight (or volume) of a hydrogel.

Examples of hydrogels include synthetic polymers such as polyhydroxyethyl methacrylate, and chemically or physically crosslinked polyvinylalcohol, polyacrylamide, poly(N-vinyl pyrolidone), polyethylene oxide,and hydrolysed polyacrylonitrile. Examples of hydrogels which areorganic polymers include covalent or ionically crosslinkedpolysaccharide-based hydrogels such as the polyvalent metal salts ofalginate, pectin, carboxymethyl cellulose, heparin, hyaluronate andhydrogels from chitin, chitosan, pullulan, gellan and xanthan. Theparticular hydrogels used in our experiment were a cellulose compound(i.e. hydroxypropylmethylcellulose [HPMC]) and a high molecular weighthyaluronic acid (HA).

A drug delivery system within the scope of the present invention can beformulated with particles of an active agent dispersed within abiodegradable polymer. Without being bound by theory, it is believedthat the release of the active agent can be achieved by erosion of thebiodegradable polymer matrix and by diffusion of the particulate agentinto an ocular fluid, e.g., the vitreous, with subsequent dissolution ofthe polymer matrix and release of the active agent. Factors whichinfluence the release kinetics of active agent from the implant caninclude such characteristics as the size and shape of the implant, thesize of the active agent particles, the solubility of the active agent,the ratio of active agent to polymer(s), the method of manufacture, thesurface area exposed, the density of the implant and the erosion rate ofthe polymer(s)

The selection of the biodegradable polymer used can vary with thedesired release kinetics, patient tolerance, the nature of the diseaseto be treated, and the like. Polymer characteristics that are consideredinclude, but are not limited to, the biocompatibility andbiodegradability at the site of implantation, compatibility with theactive agent of interest, and processing temperatures. The biodegradablepolymer matrix usually comprises at least about 10, at least about 20,at least about 30, at least about 40, at least about 50, at least about60, at least about 70, at least about 80, or at least about 90 weightpercent of the implant. In one variation, the biodegradable polymermatrix comprises about 40% to 50% by weight of the drug delivery system.

Biodegradable polymers which can be used include, but are not limitedto, polymers made of monomers such as organic esters or ethers, whichwhen degraded result in physiologically acceptable degradation products.Anhydrides, amides, orthoesters, or the like, by themselves or incombination with other monomers, may also be used. The polymers aregenerally condensation polymers. The polymers can be crosslinked ornon-crosslinked.

Polypeptide (PLA) polymers exist in 2 chemical forms, poly(L-lactide)and poly(D,L-lactide). The pure poly(L-lactide) is regioregular andtherefore is also highly crystalline, therefore degrades in vivo at avery slow rate. The poly(D,L-lactide) is regiorandom which leads to morerapid degradation in vivo. Therefore a PLA polymer which is a mixture ofpredominantly poly(L-lactide) polymer, the remainder being apoly(D-lactide) polymer will degrade in vivo at a rate slower that a PLApolymer which is predominantly poly(D-lactide) polymer. A PLGA is aco-polymer that combines poly(D,L-lactide) with poly(glycolide) invarious possible ratios. The higher the glycolide content in a PLGA thefaster the polymer degradation.

The release rate of the active agent can depend at least in part on therate of degradation of the polymer backbone component or componentsmaking up the biodegradable polymer matrix. For example, condensationpolymers may be degraded by hydrolysis (among other mechanisms) andtherefore any change in the composition of the implant that enhanceswater uptake by the implant will likely increase the rate of hydrolysis,thereby increasing the rate of polymer degradation and erosion, and thusincreasing the rate of active agent release. The release rate of theactive agent can also be influenced by the crystallinity of the activeagent, the pH in the implant and the pH at interfaces.

The release kinetics of the drug delivery systems of the presentinvention can be dependent in part on the surface area of the drugdelivery systems. A larger surface area exposes more polymer and activeagent to ocular fluid, causing faster erosion of the polymer anddissolution of the active agent particles in the fluid.

The drug delivery systems may include a therapeutic agent mixed with ordispersed within a biodegradable polymer. The drug delivery systemscompositions can vary according to the preferred drug release profile,the particular active agent used, the ocular condition being treated,and the medical history of the patient. Therapeutic agents which can beused in our drug delivery systems include, but are not limited to(either by itself in a drug delivery system within the scope of thepresent invention or in combination with another therapeutic agent):ace-inhibitors, endogenous cytokines, agents that influence basementmembrane, agents that influence the growth of endothelial cells,adrenergic agonists or blockers, cholinergic agonists or blockers,aldose reductase inhibitors, analgesics, anesthetics, antiallergics,anti-inflammatory agents, antihypertensives, pressors, antibacterials,antivirals, antifungals, antiprotozoals, anti-infectives, antitumoragents, antimetabolites, antiangiogenic agents, tyrosine kinaseinhibitors, antibiotics such as aminoglycosides such as gentamycin,kanamycin, neomycin, and vancomycin; amphenicols such aschloramphenicol; cephalosporins, such as cefazolin HO; penicillins suchas ampicillin, carbenicillin, oxycillin, methicillin; lincosamides suchas lincomycin; polypeptide antibiotics such as polymixin and bacitracin:tetracyclines such as tetracycline; quinolones such as ciproflaxin,etc.; sulfonamides such as chloramine T; and sulfones such as sulfanilicacid as the hydrophilic entity, anti-viral drugs, e.g. acyclovir,gancyclovir, vidarabine, azidothymidine, azathioprine, dideoxyinosine,dideoxycytosine, dexamethasone, ciproflaxin, water soluble antibiotics,such as acyclovir, gancyclovir, vidarabine, azidothymidine,dideoxyinosine, dideoxycytosine; epinephrine; isoflurphate; adriamycin;bleomycin; mitomycin; ara-C; actinomycin D; scopolamine; and the like,analgesics, such as codeine, morphine, keterolac, naproxen, etc., ananesthetic, e.g. lidocaine; β-adrenergic blocker or β-adrenergicagonist, e.g. ephidrine, epinephrine, etc.; aldose reductase inhibitor,e.g. epalrestat, ponalrestat, sorbinil, tolrestat; antiallergic, e.g.cromolyn, beclomethasone, dexamethasone, and flunisolide; colchicine,anihelminthic agents, e.g. ivermectin and suramin sodium; antiamebicagents, e.g. chloroquine and chlortetracycline; and antifungal agents,e.g. amphotericin, etc., anti-angiogenesis compounds such as anecortaveacetate, retinoids such as Tazarotene, anti-glaucoma agents, such asbrimonidine (Alphagan and Alphagan P), acetozolamide, bimatoprost(Lumigan), timolol, mebefunolol; memantine, latanoprost (Xalatan);alpha-2 adrenergic receptor agonists; 2-methoxyestradiol;anti-neoplastics, such as vinblastine, vincristine, interferons; alpha,beta and gamma, antimetabolites, such as folic acid analogs, purineanalogs, and pyrimidine analogs; immunosuppressants such as azathiprine,cyclosporine and mizoribine; miotic agents, such as carbachol, mydriaticagents such as atropine, protease inhibitors such as aprotinin,camostat, gabexate, vasodilators such as bradykinin, and various growthfactors, such epidermal growth factor, basic fibroblast growth factor,nerve growth factors, carbonic anhydrase inhibitors, and the like.

Pharmaceutical compositions of an anti-complement antibody may beprepared for storage as a lyophilized formulation or aqueous solution bymixing the polypeptide having the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients, or stabilizerstypically employed in the art.

The composition should contain a sufficient amount of active ingredientto achieve the desired effect (referred to herein as the “effectiveamount” as can be readily determined by a person skilled in the art. Ingeneral, the solubility of the active ingredient in water and theconcentration of the active ingredient needed in the tissue, guide theamount and rate of release of the agent. Compositions for systemicadministration will require a different “effective amount” compared tocompositions for direct injection into the eye or retina.

A person of ordinary skill in the art can easily determine anappropriate dosage to administer to a subject without undueexperimentation. Typically, a physician will determine the actual dosagethat will be most suitable for an individual subject based upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of the compound,the age, body weight, general health, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the individual undergoing therapy. Todetermine a suitable dose, the physician or veterinarian could startdoses levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. Similarly, the number of administrations of thecompositions described herein to achieve the desired effect may also bedetermined without undue experimentation. This is considered to bewithin the skill of the artisan and one can review the existingliterature on a specific agent to determine optimal dosing.

In some embodiments, the composition is administered in the form of aliquid (e.g., drop or spray) or gel suspension. Alternatively, thecomposition is applied to the eye via liposomes or infused into the tearfilm via a pump-catheter system. Further embodiments embrace acontinuous or selective-release device, for example, membranes such as,but not limited to, those employed in the OCUSERT System (Alza Corp.,Palo Alto, Calif.) In an alternative embodiment, the p53 activator iscontained within, carried by, or attached to a contact lens, which isplaced on the eye. Still other embodiments embrace the use of theposition within a swab or sponge, which is applied to the ocularsurface.

In some cases, the composition further comprises a pharmaceuticallyacceptable carrier, e.g., a pharmaceutically acceptable salt. Suitableocular formulation excipients include FDA approved ophthalmicexcipients, e.g., emulsions, solutions, solution drops, suspensions, andsuspension drops. Other suitable classifications include gels,ointments, and inserts/implants.

Exemplary excipients for use in optimizing ocular formulations includealcohol, castor oil, glycerin, polyoxyl 35 castor oil, Tyloxapol,polyethylene glycol 8000 (PEG-8000), ethanol, glycerin, cremaphor,propylene glycol (pG), polypropylene glycol (ppG), and polysorbate 80.In some cases, citrate buffer and sodium hydroxide are included toadjust pH.

Preferably, the compositions are delivered by topical, intravitreal,intraocular, subretinal, or systemic administration. For example, thecompositions are administered by intraocular injection or subretinalinjection. The compositions may also be delivered systemically.Antibodies have been previously shown to be successfully administeredand delivered via systemic delivery, such as anti-C5 antibody for thetreatment of wet age-related macular degeneration (AMD). Althoughsystemic administration of an anti-immune response therapeutic may haveadverse side effects, such as increased occurrence of infections, therehas been no evidence to date that shows that systemic administration ofanti-C5 antibody caused sufficient suppression of the immune system toincrease occurrence of infections.

As described in detail below, retinal detachment is accompanied byapoptosis in the retina. Specifically, photoreceptor cell death wasdetected in retinas after detachment. Furthermore, electroretinographywas used to assess photoreceptor function after retinal detachment. Theexamples below demonstrate that statistically significant decreases inboth a-wave and b-waves were detected by electroretinography afterretinal detachment. Thus, photoreceptor function decreases as a resultof the loss and/or death of photoreceptor cells.

Also as described in detail below, components of the alternativecomplement pathway have increased activity after retinal detachment.Increases in expression at both the RNA the protein level of alternativecomplement pathway components were detected after retinal detachment inboth mouse models of RD and human patients suffering from RD. Therefore,the alternative complement pathway plays a critical role in retinaldetachment.

As described in detail below, inactivation of components of thealternative complement pathway prevent photoreceptor cell death afterretinal detachment. For example, inactivation of Fb or C3 was shown toreduce photoreceptor cell death. The effects of inactivation ofalternative complement pathway components were also examined in mousemodels of retinal detachment and human patients with retinal detachment.

Example 1 Model of Retinal Detachment

Animal models of retinal detachment (RD) provide an understanding of thecellular mechanisms that facilitate photoreceptor cell death (Lewis G.P. et al., Eye (Lond), 2002). A mouse model of RD was utilized thatprovides a systematic and controlled system and allows the advantage ofthe genetic manipulation possible in mice (Matsumoto, H. et al., J VisExp, 2013, (79)). Briefly, RD was created in the right eye of adult mice(8-12 weeks), as previously reported (Hisatomi, T. et al., Am J Pathol,2001, 158:1271-1278), with minor modification. Briefly, mice wereanesthetized using a mixture of ketamine (80 mg/kg) and xylazine (8mg/kg). Deep anesthesia was confirmed by a toe pinch test. One drop ofproparacaine hydrochloride (0.5%) (Akorn, 17478-263-12) was administeredto each eye. A self-sealing scleral incision was made using a 30 Gneedle. Next, an anterior chamber puncture was performed from the corneato reduce intraocular pressure. A 33 G needle attached to a Hamilton 10μl syringe was inserted into the subretinal space, and 4 μl sodiumhyaluronate (Provisc®, Alcon) was gently injected to enlarge the RD(fundus Image of a retinal detachment; FIG. 7A) (Matsumoto, H. et al., JVis Exp, 2013, (79)). After the injection, glue (Webglue™) was put onthe scleral wound and the conjunctiva reattached to the originalposition. Photoreceptor apoptosis was assessed (12-72 hours postdetachment) by identifying TUNEL positive cells in cross sections (FIGS.7C and D). Maximal photoreceptor cell death in response to RD was foundto occur 24 hours post injury in this model (FIG. 7C). ERG analysisallowed for the non-invasive assessment of neural responses that can beused to objectively evaluate retinal function on a layer-by-layer basisfollowing light stimulation (Weymouth A. E., et al., Prog Retin Eye Res,2008, 27:1-44). RD results in a significant loss of retinal function (a-and b-wave) as demonstrated by ERG analysis. The levels of complementfactor-B (FB) were assessed, a key component of the alternative pathway,in the vitreous of patients with and without (macular hole) RD.

Example 2 Isolation of the ONL Using Laser Capture Micro-Dissection

Eyes were enucleated 24 hours post RD then placed in OCT compound(Tissue Tek, 4583, Torrance, Calif.), and quickly frozen by submergingin a beaker of isopropanol chilled by dry ice. Between 16 and 18sections cut at 30 μm were placed onto a frame slide (leica 11505190,Germany) under RNAse free conditions then allowed to air dry for 10minutes. All reagents were made using nuclease free water (Ambion,AM9932, Carlsbad, Calif.). The sections on the frame slides were fixedusing a graded series of EtOH (Sigma, 459836-1L, St. Louis, Mo.)consisting of incubation in 50% EtOH for 1 minute, 75% EtOH for 1.5minutes, and water for 1 minute. To identify the cell layers the slideswere dipped in 0.1% toluidine blue solution (Fluka, 89640-5G, St. Louis,Mo.) followed by 2 rinses in water for 15 seconds each, 75% EtOH for 30seconds, and water for 15 seconds. Sections were dried in room air thenthe photoreceptors were isolated using laser capture micro-dissection(Leica, LMD 7000, Germany). Samples were collected in RNAlater® solution(Ambion, AM7022, Carlsbad, Calif.) and stored at −80° C. until RT-PCRwas performed.

Example 3 Alternative Complement Pathway in RD

Increased activity of the alternative complement pathway was correlatedwith retinal detachment. In this Example, gene expression and proteinlevels of alternative complement pathway components were assessed in themouse model of RD.

To define the role of alternative complement pathway in RD a mouse modelthat allowed for the genetic manipulation possible in mice was utilized(Matsumoto, et al. J Vis Exp. 2013). In this model a sustained RD iscreated by a sub-retinal injection of sodium hylaluronate (Provise),resulting in approximately 60% of the retina becoming detached (FIG. 1B)(Matsumoto, et al. J Vis Exp, 2013). Photoreceptor apoptosis is assessedfrom 12-72 hours post detachment by identifying TUNEL positive cells inthe outer nuclear layer (ONL) (FIGS. 7A-D). The peak amount of celldeath occurs at 24 hours post-detachment (FIGS. 7C and D). Fb expressionin the retinas of mice with or without RD was assessed and foundsignificant up-regulation from 12-48 hours after detachment, peaking at24 hours (FIGS. 1C and D). Key activators for the lectin (Masp2) andclassical (C1q) complement pathways were also assessed and in both casesregulation was not as significant as the alternative pathway but therewere some time points with minor, yet significant, changes (FIG. 8).

Cd55, a regulatory protein of the alternative pathway, is suppressed inthe photoreceptors of RD mice. Soluble and cell bound regulators ofcomplement help to protect healthy host tissue from self-recognition,providing protection from erroneous activation (Nesargikar, et al. Eur JMicrobiol Immunol (Bp) 2, 103. June, 2012; Kemper et al. Clin ExpImmunol 124, 180. May, 2001; Harris, et al. Biochem J 341 (Pt 3), 821Aug. 1, 1999; Hamilton, et al. Blood 76, 2572. Dec. 15, 1990). However,damaged or diseased host cells have been shown to down-regulate membranebound regulators of complement, allowing for their targeted clearance(Suzuki et al. J Immunol 191, 4431. Oct. 15, 2013; Banadakoppa et al.Cell Biol Int 36, 901. Oct. 1, 2012; Gustafsson et al. Virology 405,474. Sep. 30, 2010. Cd55, a key regulator of the alternative pathway(Harris, et al. Biochem J 341 (Pt 3), 821 Aug. 1, 1999), was found to besignificantly down-regulated in the detached retina (FIG. 1C) makingthese cells intrinsically more prone to targeted cell death. To confirmthat the decline in Cd55 is specific to the photoreceptors the ONL wasisolated using laser capture micro-dissection (FIGS. 9A and B) andassessed gene expression by real-time PCR for Cd55.

RNA was isolated using an RNeasy micro kit (Qiagen, 74004, Valencia,Calif.) for LCM samples or an RNeasy mini kit (Qiagen, 74106, Valencia,Calif.) for whole retina. Total RNA was measured using a nanodropspectrophotometer (Thermo Scientific, Nanodrop 2000, Waltham, Mass.)then each sample was normalized prior to transcribing cDNA. cDNA wastranscribed using superscript III (Invitrogen, 18080-044, Carlsbad,Calif.) then one microliter of cDNA was used for each RTPCR reaction.Primers to Fb (Life Technologies, Mm004333909), C1q (Life Technologies,Mm00432142), MASP2 (Life Technologies, Mm00521963), Cd55 (LifeTechnologies Mm00438377), and Cd59a (Life Technologies, Mm00483149) wereused for whole retina and combined with Taqman universal PCR master mix(Life technologies, 4304437). Primers for Cd55 using photoreceptorsisolated by LCM were obtained through Integrated DNA Technologies usingtheir online Primer Quest design tool and importing the NCBI ID numberfor the sequence (Forward 5′-TGTAAGCAGAATCGCCACAGAGGT-3′ (SEQ ID NO: 5)and Reverse 5′-GTGAGCTTCCACTGCAGGTTTGTT-3′ (SEQ ID NO: 6)). RTPCR wasrun in triplicate and the average of each CT value used for analysis.All CT values were normalized to beta actin from each sample as aninternal control. Final values were determined using the MET method.

Cd55 expression levels in the photoreceptors were significantly reduced(74.6%±5.262) in response to RD (FIG. 1E). In RD the photoreceptorsappear to be highly susceptible to alternative pathway mediated celldeath due, in part, to down-regulation of the complement regulator Cd55.

Example 4 Alternative Complement Pathway in Human RD

In this Example, the role of the alternative complement pathway inretinal detachment was confirmed in human patients. Patient vitreoussamples were collected and processed 1-14 days after diagnosis ofsymptoms or detachment. Control vitreous samples were obtained frompatients with macular hole, which is a natural control for retinaldetachment. Undiluted vitreous (0.3 to 1.0 mL volume) was obtained frompatients during standard three-port pars plana vitrectomy under directvisual control by aspirating liquefied vitreous from the center of thevitreous cavity with a syringe before starting the infusion. Vitreouswas obtained from 9 patients with varying degrees of retinal detachment(RD) and 4 patients that had a macular hole with no RD (control samples)(FIG. 5). Samples were kept on ice during the time of surgery thenimmediately moved to −80° C. for storage. In order to separate thesoluble protein from the collagenous matrix the samples were thawed onice then spun at 12,000 RPM for 15 minutes at 4° C. (J. Yu et al.,Proteomics 8, 3667 (September, 2008). The supernatant was collected,aliquots taken, and stored at −80° C. Each aliquot was thawed not morethan once for use in the ELISA assays.

Protein levels of factor-b (Fb), a key component in the alternativecomplement pathway, were assessed by ELISA. All human ELISA measurementswere performed using 5 μl of undiluted vitreous, isolated as describedabove. All ELISAs were performed following kit instructions and measuredusing a Molecular Devices Spectramax M3 plate reader. Standard curveswere generated using the standard reagents provided in the kit thensample values determined automatically using Softmax pro software. HumanELISA kits that were used include Fb (Novatein, BG-HUM10501), Fd (Abcam,ab99969), C5 (Abcam, 125963), and C3 (Abcam, ab108822).

Factor-B was significantly up regulated in RD patients, indicatingalternative pathway activation (FIG. 1A). Interestingly, there was nosignificant change in several other key complement proteins; includingFactor D, C5, and C3 (FIGS. 6C-E). This is in line with previous studiesthat have shown that there are key regulatory proteins that undergotranscriptional control to modulate the activity of the complementpathways (Hecker et al. Hum Mol Genet 19, 209. Jan. 1, 2010; Nielsen, etal. Apmis 100, 1053. December, 1992; Suankratay, et al. Clin Exp Immunol117, 442. September, 1999). For the alternative complement pathway FBhas been described as a key effector molecule responsible for pathwayactivation (Hecker et al. Hum Mol Genet 19, 209. Jan. 1, 2010). Howeverinhibition of any component of the pathway is sufficient to blockcomplement activation.

Example 5 Complement Pathway Inactivation Prevents Photoreceptor CellDeath

The role of the alternative complement pathway was analyzed inphotoreceptor cell death during retinal detachment. Transgenic knockoutmice were utilized to assess the effect of alternative complementpathway inactivation on retinal cell death after retinal detachment.

Retinal detachment was induced in mice lacking alternative complementcomponent Fb (Fb^(−/−)) and their wild-type (WT) littermates(age-matched control), as described in Example 1. Twenty-four hourspost-injury, retinas were isolated and cross-sections were prepared forstaining with DAPI (a nuclear marker) and TUNEL (a cell death marker).The mid-point of the retinal arc between the optic nerve and the edge ofthe retina was quantified for TUNEL positive cells. Representativestaining of the retinal cross-sections are shown in FIG. 4A).Quantification of the cross sections (FIG. 3B) showed that mice lackinga functional alternative complement cascade were significantly protectedfrom photoreceptor cell death. This data implicates the alternativecomplement system in initiating photoreceptor cell death. It alsodemonstrates that neutralizing or inhibiting the alternative complementcascade can be sufficient to protect from photoreceptor degeneration.

The experiment was also performed in mice lacking the complementcomponent C3. Photoreceptor cell death was assessed 24 hourspost-retinal detachment by TUNEL staining of retinal cross-sections. Asshown in FIG. 2F, loss of the complement component-3 protects mice fromphotoreceptor cell death resulting from retinal detachment.

C3 neutralizing antibody: In order to dampen complement activation aneutralizing antibody was injected against the central C3 proteinrequired for complement amplification. Prior to in vivo injection theazide was removed from the C3 antibody (Abcam, ab11862) using an Abcampurification kit (ab102784) following kit instructions. Briefly, 200 μlof C3 antibody was used for starting material and incubated in theprovided resin containing 20 μl of 10× binding buffer for 1 hour at roomtemperature with gentle agitation. The resin was then spun to removeunbound antibody and washed with the provided wash buffer. The antibodywas eluted with 100 μl of elution buffer then 25 μl of neutralizer wasadded. The elution procedure was repeated 3 times using separatecollecting tubes. Only the first tube was used for in vivo injectionscorresponding to 0.1 mg/ml. The model of retinal detachment wasperformed precisely as described above with one modification: Prior toinjecting the provisc 1 μl of the purified C3 antibody (0.1 μg) or IgG2aisotype control (Life Technologies, 02-9688) was injected into thesub-retinal space through the scleral tunnel. The eyes were enucleated24 hours after RD and processed for TUNEL labeling as described earlier.

Fd neutralizing antibody: To dampen the alternative pathway an antibodywas injected that binds to Fd (R&D systems, MAB5430) prior to creatingRD. The antibody did not contain azide therefore was resuspended in PBSto a final concentration of 0.5 mg/ml, aliquots taken, and stored in−20° C. The antibody was not thawed more than once for injection. Themodel of retinal detachment was performed precisely as described abovewith one modification: Prior to injecting the Provisc® 1 μl of thepurified Fd antibody (0.5 μg) or IgG1 isotype control (LifeTechnologies, R100) was injected into the sub-retinal space through thescleral tunnel into the sub-retinal space. The eyes were enucleated 24hours after RD and processed for TUNEL labeling as described earlier.

Conversely, restoration of the complement pathway ablates the protectionprovided by inactivation of the complement cascade. C3 knockout micewith retinal detachments were administered cobra venom factor (CVF) orPBS (control). Cobra venom factor is a protein that is a mimetic fornormal endogenous C3/C3b and therefore restores the complement cascadein the C3 knockout mice. Mice without C3 and injected PBS were protectedfrom photoreceptor cell death. However, mice injected with CVF andrestoration of the complement cascade exhibited a significantup-regulation of photoreceptor cell death as a result of retinaldetachment. This data demonstrates that the complement systemfacilitates photoreceptor cell death in response to injury (retinaldetachment). Inhibition of this pathway protects photoreceptors frominjury-mediated cell death and subsequent retinal degeneration.

Re-activation of the complement system in C3^(−/−) mice: To re-activatecomplement in C3^(−/−) mice 1 μl (1.1 mg) of cobra venom factor (CVF)(quidel, A600) into the sub-retinal space was injected prior to theProvisc® injection. When introduced into the blood stream CVF activatescomplement, bypassing the need for endogenous C3. The rest of the modelof retinal detachment was performed precisely as described above. Theeyes were enucleated 24 hours after RD and processed for TUNEL labelingas described earlier.

Example 6 Alternative Pathway Deficient Mice are Protected from RDAssociated Photoreceptor Cell Death

Photoreceptor cell death in response to RD was next assessed in aC3^(−/−) mouse, which lacks the central C3 protein required for allthree complement pathways (Ricklin, et al. Nat Immunol 11, 785.September, 2010). Photoreceptor cell death was quantified at 24 hourspost detachment, the peak of cell death. C3^(−/−) mice had a reductionin the number of TUNEL positive cells compared to their age and strainmatched controls (FIGS. 2A and B). To further define the role of C3 inRD, C57B16 (control wild type) mice were given injections of an antibody(Ab) against C3 in the sub-retinal space at the time of detachment.Quantitation of TUNEL labeling revealed that administration of an antiC3 Ab significantly protected the mice from photoreceptor cell death inresponse to RD (FIGS. 2C and D). Conversely, complement was re-activatedin C3^(−/−) mice by introducing cobra venom factor (CVF) which is astable functional analog to C3b (Krishnan et al. Structure 17, 611. Apr.15, 2009). This bypasses the need for C3 by replacing the C3 cleavageproduct, C3b, required for the alternative pathway proteolytic cascadeto continue. The reactivation of the complement system in C3^(−/−) miceusing CVF increased photoreceptor cell death after RD (FIGS. 2E and F).Taken together these results strongly implicate the complement system asa driving force in promoting the early photoreceptor cell deathassociated with RD.

The alternative complement pathway remains in a primed state throughconstant tick-over of the central alternative pathway C3 proteinallowing for continuous probing within the retinal microenvironment (andCNS) for the identification of cells that are damaged or dying;distinguishing the alternative pathway from the lectin and classicalpathways (Ricklin, et al. Nat Immunol 11, 785. September, 2010; Bexborn,et al. Mol Immunol 45, 2370. April, 2008). To determine the role of thealternative pathway in photoreceptor cell death during RD mice that lackFb, an essential rate limiting protein required for alternative pathwayactivation after C3 cleavage were tested (Hecker et al. Hum Mol Genet19, 209. Jan. 1, 2010). Mice deficient in the alternative complementpathway (Fb^(−/−)) had a substantial decrease in photoreceptor celldeath 24 hours post RD (FIGS. 3A and B). Complement factor-d (Fd) is aserine protease, which cleaves Fb once bound to C3b resulting in theassembly of the alternative pathways C3-convertase (Ricklin, et al. NatImmunol 11, 785. September, 2010). To pharmacologically block thealternative complement pathway in RD an antibody against Fd was injectedinto the sub-retinal space of C57BL6 control mice at the time ofdetachment. Neutralization of Fd resulted in a reduction in the amountof photoreceptor cell death compared to isotype matched controls (FIGS.3C and D). Notably, mice deficient in either the lectin (Mbl A/C^(−/−))or classical (C1q^(−/−)) pathways did not confer protection againstcomplement mediated photoreceptor cell death (FIGS. 10A-D). This datadirectly implicates the alternative complement pathway and not thelectin or classical in mediating photoreceptor cell death in response toRD.

Example 7 Efficacy of Anti-Complement Agents as Therapeutics

Retinas are detached as described in Example 1. At the time of retinaldetachment, the mice are administered either inhibitor or a salinecontrol by intraocular injection, subretinal injection, or systemicadministration. After 24 hours, retinal cross-sections are prepared andstained by TUNEL to assess photoreceptor cell death, as described inExample 5. Additional timepoints (i.e., at 12 hours, 36 hours and 48hours after retinal detachment) are assessed to verify the efficacy andoptimal timing of administration.

Photoreceptor cell viability after treatment is assessed byelectroretinography to determine photoreceptor cell death and extent ofpreservation of photoreceptors and vision. Retinal cross-sections areprepared, and stained with cell death markers (such as TUNEL) todetermine increased or reduced photoreceptor cell death. Optomotorvisual tests are performed to test preservation of vision.

Example 8 Retinal Hypoxia Leads to Alternative Pathway Activation andCell Death in RD

Photoreceptor cells are one of the most highly metabolic cell types inthe body (Linsenmeier, et al. Invest Ophthalmol Vis Sci 41, 3117.September, 2000). Yet with such high metabolic demand they are notpermeated with a vascular network, deriving 90% of their nutrients andoxygen by diffusion from the vascular bed of thechoroid/choriocapillaris (Linsenmeier, et al. Invest Ophthalmol Vis Sci41, 3117. September, 2000; Luo, et al. Elife 2, e00324. 2013). When RDoccurs it physically separates the photoreceptor cells from the RPE anddistances them from the choroid thereby compromising access to nutrientsand oxygen. Several seminal studies have shown that the deprivation ofoxygen (hypoxia) is a leading cause of photoreceptor cell death (Lewis,et al. Mol Neurobiol 28, 159. October, 2003; Lewis, et al. Am JOphthalmol 137, 1085. June, 2004) in RD. Studies were carried out todetermine whether hypoxia could facilitate alternative complementpathway activation and photoreceptor cell death in response to RD.

To define global hypoxia in photoreceptors in response to RD mice wereinjected 22.5-hours after detachment intraperitoneally with a marker ofhypoxia, Hypoxyprobe™ Hypoxyprobe™ is a thiol binding probe that onlybinds to cells with an oxygen concentration less than 14 μmol which canbe visualized through diaminobenzadine (DAB) amplification. In retinalcross sections the detached photoreceptors stained strongly, indicatinghypoxia, whereas in the attached regions of the same cross section nostaining could be observed (FIG. 4A).

To obtain a more precise reading of the retinal oxygen concentration invivo a glass oxygen microsensor that contains a silicone membrane wasused allowing for the diffusion of oxygen into an oxygen-reducingcathode. In vivo oxygen measurements were performed using a 50 μmdiameter glass-electrode-oxygen sensor (Unisense A/S, Denmark). Thisprobe has a linear response between 0-1 atm pO₂ ranges, negligibleoxygen consumption (10-16 mol/sec) and fast time response. The cathodeis polarized against an internal Ag/AgCl anode that results in rapid,0.3 second, in vivo oxygen measurements. Prior to its use, the sensorwas thoroughly pre-polarized for 24 hours and was calibrated using twopoints: maximum oxygen saturation using 0.9% saline at 37° C. undercontinuous air agitation, and zero oxygen calibration using sodiumascorbate solution. A linear fit was calculated from the two calibrationpoints and implemented into the data acquisition software. Sampling ratewas adjusted to 1 Hz (Nyquist-Shannon sampling theorem) and measurementswere acquired and stored in a portable computer for later processing.

Placing the probe into the attached retina using a stereotaxic frame itwas defined an average oxygen concentration of 47.64±3.346 μmol comparedto the contralateral, detached, retina with an average oxygenconcentration of 17.14±5.031 μmol (FIG. 4B). To assess if the hypoxicstate of the photoreceptors facilitates alternative pathway mediatedcell death RD in mice housed in room air (approximately 21% oxygen) orhoused in a chamber maintained at 75% oxygen were analyzed. After 24hours, eyes were enucleated and TUNEL labeling performed. Quantitationof TUNEL positive cells in the ONL revealed a significant reduction incell death within the group of mice maintained at 75% oxygen (FIGS. 4Cand D). Importantly, the mice kept in 75% oxygen had significantly lessFb expression, alternative pathway activation, than their room aircounterparts (FIG. 4E) indicating an oxygen dependent regulation of Fb.

Mice were anesthetized by using a mixture of ketamine (80 mg/kg) andxylazine (8 mg/kg) administered via an intra-peritoneal (IP) injection.Adequate sedation was confirmed by toe pinch. One drop of proparacainehydrochloride (0.5%) (Akorn, 17478-263-12) was administered to each eye.A stereotaxic frame (Leica 39463001) with Cunningham mouse adaptor(Leica 39462950) was used to immobilize the head of the mouse. A 32gauge needle was then used to create a tunnel into the subretinal space.A manual (x,y,z) microstage, adapted with the oxygen sensor, was used togently guide the probe into the subretinal space adjacent to thedetached retina and measurements were performed for several seconds,until oxygen readings were stabilized. Oxygen measurements in thecontralateral non-detached eyes served as internal controls and naïvemice served as control reference. During the experimental time frame theprobe was retested for accuracy in the calibration solutions and foundto be within range. The mice were euthanized by spinal dislocation afterthe final reading.

Example 9 Detecting Hypoxic Regions in the Retina

Hypoxic areas of the retina were identified 24 hours after detachmentwith the use of the Hypoxyprobe™-1 Plus kit (HPI, HP2-100). Hypoxyprobe™(pimonidazole hydrochloride) is a substituted 2-nitoimidazole that formsadducts with thiol containing proteins in only those cells that have anoxygen concentration less than 14 μmol (e.g. hypoxic cells). Retinaldetachments were made as described above and 22.5 hours later mice wereanesthetized with a mixture of ketamine (80 mg/kg) and xylazine (6mg/kg) then injected with Hypoxyprobe™ (2 mg in a volume of 100 μl)directly into the left ventricle. Mice were kept on a heating padmaintained at 37° C. and monitored for 90 minutes to allow completebinding of the probe to hypoxic tissues. The eyes were enucleated andfrozen in OCT that had been chilled in dry ice. Blocks were sectioned at20 μm then fixed in 4% PFA for 20 minutes. The endogenous peroxidasepresent in the tissue was quenched using 3% H₂O₂ for 5 minutes.Non-specific binding was blocked using 5% fetal calf serum/0.5% tritonx/0.3% bovine serum albumin. Samples were then incubated with a FITCconjugated mouse IgG1 that binds to pimonidizole (Hypoxyprobe™) at adilution of 1:100. The FITC was amplified using rabbit anti-FITCconjugated with horseradish peroxidase at a dilution of 1:75 followed bythe addition of a brown DAB chromagen. To identify the retinal layersslides were dipped in haematoxylin (Sigma, H9627) for 1 minute thenwashed 3 times in PBS and coverslipped. Binding of the probe wascompared on retinal cross sections where 50% of the retina was attachedand the other 50% detached.

Example 10 Supplementing Oxygen after Retinal Detachment

Retinal detachments were performed as described above then the mice werekept either in the animal holding facility (≈21% O₂) or immediatelyplaced into an oxygen chamber maintained at constant 75% O₂ (Biospherixmodel 110). After 24 hours the mice were anesthetized under a heavy doseof avertin (2,2,2 tribromo ethanol, Sigma, T4, 840-2) at a workingconcentration of 10 mg/ml and injected at a dose of 0.25-0.5 mg/gramintraperitoneally. In one group the eyes were enucleated and frozen inOCT compound using isopropanol chilled by dry ice. The eyes were thenprocessed for TUNEL labeling and quantified as described above. In theother group the retina was removed and immediately flash frozen inliquid nitrogen for RNA extraction. All eyes were stored at −80° C.

Example 11 Clinical Trial for Anti-Complement Therapeutics for TreatingRD

A clinical trial is performed to determine the efficacy ofanti-complement therapeutics in human patients suffering from retinaldetachment. Preferably, the anti-complement inhibitors are administeredto subjects as soon as possible once the retinal detachment was evident.The patients are diagnosed with retinal detachment using fundus imagery,ophthalmoscope, or ultrasonography. A clinical professional or physicianmay also diagnose retinal detachment by identifying at least one symptomassociated with retinal detachment. Other patient cohorts, such as thosesuffering from various ocular disorders with an increased risk ofretinal detachment are also assessed.

Anti-complement inhibitors are administered to the subjectssystemically, by intra-ocular injection or sub-retinal injection. Theinhibitors are administered within 12 hours, 24 hours, or 48 hours ofthe diagnosis of retinal detachment. Placebos are administered via thesame delivery methods as a control.

Assessment of the efficacy of treatment is determined in the subjectsbefore and after delivery of the anti-complement inhibitor or theplacebo. Efficacy of treatment is determined by evaluating photoreceptorcell activity and vision. Electroretinography is used to measurephotoreceptor cell activity. Electrodes are placed on the cornea and/oron the skin surrounding the eye. During recording, the patient's eyesare exposed to standardized light stimulus (flash or pattern stimulus),and the resulting signal is displayed and analyzed.

Spectral domain optical coherence tomography (SD-OCT) is also used fordetailed and non-invasive evaluation of the retinal architecture invivo. SD-OCT is used to show retinal morphological changes duringretinal detachment.

Standard eye examinations are also performed to evaluate visionpreservation or loss. Visual acuity is tested using Sneller charts, totest the ability of the subject to distinguish varying sizes of lettersand/or numbers from a distance. Peripheral vision can also be testedusing visual field exams.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A composition for preserving vision or reducing vision loss in asubject, comprising an agent that inhibits or reduces alternativecomplement pathway activity.
 2. A composition for inhibiting or reducingphotoreceptor cell death in a subject, comprising an agent that inhibitsor reduces alternative complement pathway activity.
 3. The compositionof claim 1, wherein said agent inhibits or reduces the activity ofFactor D, factor B (Fb), C5 or C3.
 4. The composition of claim 1,wherein said composition is suitable for intraocular injection orsystemic administration.
 5. The composition of claim 1, wherein saidagent comprises an antibody or an antigen-binding fragment thereof, asmall molecule, a polynucleotide, or a polypeptide.
 6. The compositionof claim 1, wherein said agent comprises a serine protease inhibitor, asoluble form of a complement receptor, a humanized monoclonalanti-complement antibody or antibody fragment, a complement componentinhibitor, a nucleic acid expression vector encoding anti-complementpolypeptides, or an anaphylatoxin receptor antagonist.
 7. Thecomposition of claim 1, wherein said agent inhibits or reduces theactivity of at least one selected from factor B (Fb), C3, properdin(Factor p), factor Ba, factor Bb, factor D, C2, C2a, C3a, C3b, C5, C5a,C5b, C6, C7, C8, C9, and C5b-9.
 8. The composition of claim 1, whereinsaid agent inhibits or reduces the transcription stability, translation,modification, localization, cleavage, or function of a polynucleotide orpolypeptide encoding any one of the selected from factor B (Fb), C3,properdin (Factor p), factor Ba, factor Bb, factor D, C2, C2a, C3a, C3b,C5, C5a, C5b, C6, C7, C8, C9, and C5b-9.
 9. The composition of claim 1,wherein said agent is selected from the group consisting of cinryze,berinert, rhucin, eculizumab, pexelizumab, ofatumumab, TNX-234,compstatin/POT-4, PMX-53, rhMBL, human CD55, BCX-1470, C1-INH,SCR1/TP10, CAB-2/MLN-2222, mirococept, sCR1-sLe^(x)/TP-20, TNX-558,TA106, Neutrazumab, anti-properdin, HuMax-CD38, ARC1905, JPE-1375, andJSM-7717.
 10. The composition of claim 5, wherein said antibodycomprises a monoclonal antibody.
 11. The composition of claim 5, whereinsaid antibody comprises a chimeric antibody.
 12. The composition ofclaim 5, wherein said antibody comprises a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment or ScFv fragment.
 13. The composition ofclaim 5, wherein said antibody specifically binds to Fb or C3.
 14. Thecomposition of claim 5, wherein said antibody specifically binds to anyone selected from the group consisting of factor B (Fb), C3, properdin(Factor p), factor Ba, factor Bb, factor D, C2, C2a, C3a, C3b, C5, C5a,C5b, C6, C7, C8, C9, and C5b-9.
 15. The composition of claim 1, whereinthe subject suffers from an ocular disorder associated withcomplement-mediated photoreceptor cell death.
 16. The composition ofclaim 1, wherein the subject suffers from retinal detachment.
 17. Thecomposition of claim 1, wherein the subject suffers from trauma-inducedretinal detachment.
 18. A pharmaceutical composition comprising thecomposition of claim 1 and a pharmaceutically acceptable carrier. 19.The pharmaceutical composition of claim 18, wherein said composition issuitable for topical, intraocular, intravitreal, subretinal, or systemicadministration.
 20. A method of preserving vision or reducing visionloss in a subject comprising administering to the eye of said subjectany one of the composition of claim
 1. 21. A method of inhibiting orreducing photoreceptor cell death in a subject comprising administeringto the eye of said subject the composition of claim
 1. 22. The method ofclaim 20, wherein the subject suffers from retinal detachment.
 23. Amethod of treating or alleviating a symptom of retinal detachment in asubject comprising administering to the eye of said subject thecomposition of claim
 1. 24. The method of claim 23, wherein the retinaldetachment is induced by trauma.
 25. The method of claim 20, whereinsaid administering is by intraocular injection.
 26. The method of claim20, wherein said administering is topical, intraocular, intravitreal,subretinal, or systemic.
 27. The method of claim 23, wherein saidsymptom is selected from observing floaters in the field of vision,observing flashes of light, photoreceptor cell death, loss in peripheralvision, central vision loss, decrease in visual acuity, and blindness.28. The method of claim 23, wherein said composition is administeredwithin 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20hours, 22 hours, or 24 hours after retinal detachment.
 29. A compositionfor preserving vision or reducing vision loss in a subject, comprisingan agent that inhibits or reduces lectin complement pathway activity.30. A composition for inhibiting or reducing photoreceptor cell death ina subject, comprising an agent that inhibits or reduces lectincomplement pathway activity.
 31. The composition of claim 29 or 30,wherein said agent inhibits or reduces the activity of MASP-1, MASP-2,MASP-3, Map19, Map44, C4, C4a, C4b, C2, C2a or C2b.
 32. The compositionof claim 29, wherein said agent inhibits or reduces the transcriptionstability, translation, modification, localization, cleavage, orfunction of a polynucleotide or polypeptide encoding any one of theselected from MASP-1, MASP-2, MASP-3, Map19, Map44, C4, C4a, C4b, C2,C2a or C2b.
 33. The composition of claim 29, wherein said agentcomprises C1-inh, antithrombin, sunflower MASP inhibitors SFMI-1 orSFMI-2, or SMGI inhibitors SGMI-1 or SGMI-2. 34.-37. (canceled)